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Abstract

Aim: To develop a useful alternative approach to evaluate the gene function in hairy roots.
Methods:Arabidopsis and tobacco (wild-type or mutant) were a host for
Agrobacterium rhizogenes transformation.
Results: The hairy roots formation efficiency ranged from 53 to 98% in tobacco and 53 to 66%
in
Arabidopsis. Hairy and intact roots showed similar gene expression pattern in response to salt
and aluminum stress. Genomic polymerase chain reaction and fluorescent images showed
high rate (>80%) of co-integration of T-DNAs and uniform cell transformation without
use of any antibiotic selection. Whole processes of hairy roots were completed within
1 month after the infection of
Agrobacterium.
Conclusion: Aluminum-responsive orthologous gene function could be evaluated by
NtSTOP1-KD and
Atstop1 as a host for hairy roots transformation.

Lay abstract

Hairy roots have been used for various purposes over the last several years. In the
present study we used
Agrobacterium rhizogenes-mediated hairy roots as an alternative approach for
Agrobacterium tumefaciens transformation. We developed a simple, effective and reproducible hairy root protocol
in tobacco and
Arabidopsis. Developed hairy roots were compared with intact roots to characterize the gene response
to aluminum, NaCl stress and cellular localization of genes. Aluminum-responsive orthologous
genes function could be evaluated by using
NtSTOP1-KD and
Atstop1 as a host for hairy roots transformation.

The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.

Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.